In vitro translation and processing of cathepsin B of Schistosoma mansoni.
Cathepsin B has been implicated with a key role in schistosome metabolism. Here, we investigate the processing of preprocathepsin B to the mature enzyme in in vitro transcription and translation experiments. The cDNA sequence encoding the precursor of cathepsin B of Schistosoma mansoni was cloned in a modified RNA expression vector pSP65 and the derived RNA expressed in rabbit reticulocyte lysates. Transcripts yielded two translation products with mol. wts of 37.5 and 31.5 kd. The former, believed to be identical to preprocathepsin B is cleaved into a smaller 28 kd polypeptide, representing the mature enzyme. By using a mutated form of preprocathepsin B, in which the active site cysteine is substituted by alanine, no processing is observed. Cleavage is enhanced by the addition of exogenous bovine cathepsin B and inhibited in the presence of specific cysteine protease inhibitors, but not inhibitors of other proteases. The 31.5 kd product is presumably translated from an internal initiation codon. Our in vitro transcription and translation system provides a useful experimental basis for elucidating the processing mechanisms of preprocathepsin B conversion to the active protease.
Felleisen
R
R
Zentrum für Molekulare Biologie, University of Heidelberg, FRG.
Klinkert
M Q
MQ
D000818:Animals; D002401:Cathepsin B / Q000235:genetics*; D002403:Cathepsins / Q000235:genetics*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics*; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D012550:Schistosoma mansoni / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D002403:Cathepsins; C057831:preprocathepsin B; D002401:Cathepsin B